Fusion proteins

ABSTRACT

A DNA sequence encoding a fusion protein comprising influenza virus HA, at a site of which normally occupied by a natural antigenic epitope thereof a different antigenic epitope is provided, is incorporated in a vector which is capable of expressing the fusion protein when provided in a eucaryotic host. When in the form of a viral vector such as a recombinant vaccinia virus, the vector can be used as a vaccine. Alternatively, the fusion protein expressed by the host can be recovered and provided as a vaccine.

This is a continuation of application Ser. No. 08/116,557, filed Sep. 7,1993, now abandoned, which is a continuation of Ser. No. 07/856,806,filed Mar. 24, 1992 (abandoned), which is a continuation of 07/545,766,filed Jun. 28, 1990, now abandoned which is a continuation of Ser. No.07/012,943, filed Feb. 10, 1987, now abandoned.

This invention relates to the construction of fusion proteins.

In previous attempts to express immunogenic epitopes of foot-and-mouthdisease virus (FMDV) using vaccinia virus protein, fusions have beenmade to beta-galactosidase. High levels of fusion protein weresynthesised. However, animals vaccinated with the recombinant vacciniavirus failed to produce neutralizing antibody (Newton et al, 1986). Thereason for this poor response may be because beta-galactosidase isexpressed in the cytoplasm. We have therefore investigated whether anantigen which is expressed on the surface of a cell would elicit abetter response.

DNA sequences were constructed, each encoding a fusion proteincomprising influenza virus haemagglutinin protein (HA) and, at antigenicsite A of the HA, an epitope from part of the major FMDV antigenic site.The DNA sequences were incorporated into the vaccinia virus genome.Cells infected with the recombinant viruses not only expressed thefusion protein but did so such that the FMDV epitopes could be detectedusing anti-FMDV serum. Further, the infected cells reacted withpolyclonal anti-HA antibody. It is therefore possible to change thesequence of influenza HA yet still retain all its properties of membraneinsertion.

This approach for presenting antigenic epitopes to the immune system hasgeneral applicability. Accordingly, the present invention provides a DNAsequence encoding a fusion protein comprising influenza virus HA at asite of which normally occupied by a natural antigenic epitope thereof adifferent antigenic epitope is provided. In other words all or part of anatural antigenic epitope of HA may be replaced by a different antigenicepitope.

For expression of the fusion protein, the DNA sequence is incorporatedin an expression vector. The DNA sequence is incorporated in a vectorsuch that the vector, when provided in a eucaryotic host, is capable ofexpressing the fusion protein. A eucaryotic host is required for correctglycosylation of the HA. The vector may be a viral vector whichincorporates the DNA sequence such that the fusion protein is expressedin cells infected with the vector. A vaccine may comprise such a viralvector and a physiologically acceptable carrier or diluent. In such aninstance, the viral vector preferably is a recombinant vaccinia viruswhich incorporates the DNA sequence. Alternatively, a vaccine maycomprise the fusion protein and a physiologically acceptable carrier ordiluent.

Influenza virus HA is the most thoroughly studied integral membraneprotein with detailed information available on its three-dimensionalstructure (Wilson et al, 1981) and its antigenicity (Wiley et al, 1981).The HA genes from several influenza subtypes have been expressed in anumber of eukaryotic cells using various specific vectors (e.g. Smith etal, 1983; Gething and Sambrook, 1982). When expressed in recombinantvaccinia virus infected cells the HA is glycosylated normally andtransported to the cell surface where it can be detected by serologicalmethods.

Antigenic site A "loops out" from the surface of HA. This site can bedetermined by epitope mapping. Typically, it corresponds to HA aminoacid residues 140 to 146. The HA may correspond to any influenza virustype or subtype. Consequently, it is at HA antigenic site A for exampleat which the antigenic epitope for the fusion protein is provided. Allor part of site A may be replaced by the antigenic epitope. Typically,though, the epitope is inserted between HA residues 142 or 143 andresidue 146 with the loss of the intervening HA residues. However, theantigenic epitope may be provided at any of the other natural antigenicepitopes of HA, sites B, C, D or E.

Any antigenic epitope may be provided at the natural antigenic epitopeof HA. The epitope may be a corresponding epitope from a different typeor subtype of influenza virus as that to which the HA corresponds.Alternatively or additionally, one or more of the other naturalantigenic epitopes of HA of the same or of a different type or subtypeof influenza virus may be provided. In this way a polyvalent influenzavaccine may be formed.

More usually, however, a heterologous antigenic epitope is provided. By"heterologous" is meant an epitope which is not an epitope of aninfluenza virus. The size of the heterologous epitope usually is largerthan the portion of the HA site A which it replaces. The heterologousepitope may form part of a longer amino acid sequence. The insert mayhave from 4 to 26 amino acid residues. A heterologous antigenic epitopemay be provided with an influenza virus epitope. Two or moreheterologous antigenic epitopes may be provided. In this way, polyvalentvaccines can be presented.

The heterologous epitope may be that of a virus, bacterium or protozoan.As examples of viral epitopes, there may be mentioned those of FMDV,poliovirus, human rhinovirus and hepatitis B virus. Protozoans whoseepitopes may be provided include the malaria parasite Plasmodiumfalciparum.

The major FMDV antigenic sites correspond to amino acid residues 141 to160 and 200 to 213 of the VP1 capsid protein. Either or both of theseamino acid residues may therefore be provided, for example at site A.Alternatively parts of these sequences may be provided e.g. residues 142to 145, 146 to 151 or 142 to 151. Suitable DNA constructs incorporatingparts of the major antigenic site of FMDV type O₁ Kaufbeuren, and theircorresponding amino acid sequences as denoted by the one-letter code,are shown below together with the DNA and amino acid sequences for HA.##STR1##

The DNA sequence encoding the fusion protein can be prepared byproviding a DNA sequence encoding HA, for example as a plasmid, andmodifying this sequence by providing in the correct frame at the site ofthe DNA encoding a natural antigenic epitope of HA a DNA sequencecorresponding to the desired antigenic epitope it is wished toincorporate in the fusion protein. A vector capable of expressing thefusion protein may be prepared by incorporating the DNA sequenceencoding the fusion protein between translational start and stop signalsin a vector suitable for use in a eucaryotic host and providing apromoter for the sequence. By transforming eucaryotic host cells withsuch an expression vector, the fusion protein can be produced.

A viral vector for use as a vaccine may therefore be prepared byincorporating a DNA sequence encoding the fusion protein betweentranslational start and stop signals in the genome of a virus andproviding a promoter for the sequence. Typically, this may be achievedby:

(i) constructing a shuttle vector which incorporates, under thetranscriptional control of a promoter, a DNA sequence encoding HAbetween translational start and stop signals;

(ii) modifying the DNA sequence by providing in the correct frame at thesite of the DNA encoding a natural antigenic epitope of HA a DNAsequence corresponding to the antigenic epitope to be incorporated inthe fusion protein; and

(iii) transfecting with the shuttle vector and infecting with a virusmammalian cells such that the thus-modified DNA sequence and thepromoter are incorporated in the viral genome.

The DNA sequence and the promoter are incorporated in the viral genomeby homologous recombination. Appropriate flanking sequences of viral DNAare therefore provided on either side of the DNA sequence and promoterin the shuttle vector. The fusion protein is expressed by cells infectedwith the resultant recombinant virus.

The shuttle vector is typically a plasmid. It comprises a bacterialorigin of replication to enable steps (i) and (ii) to be carried out inbacteria, especially E. coli. The promoter is typically a viralpromoter, more preferably a promoter endogenous to the virus into thegenome of which the DNA encoding the fusion protein is to be inserted.The antigenic epitope is generally prepared by chemical synthesis and/orby cloning. Some or all of the codons of the HA antigenic epitope may beexcised from the shuttle vector prior to insertion of the DNA sequenceencoding the epitope to be incorporated in the fusion protein.

A vaccinia virus system may be used. A shuttle vector may be constructedin which the HA gene is incorporated under the transcriptional controlof a vaccinia promoter. A suitable promoter is the vaccinia p11kpromoter. The promoter and HA gene are flanked by vaccinia virus DNAwhich is not essential for virus replication. Typically, flankingsegments of the vaccinia gene for thymidine kinase (TK) are used. The HAgene is modified by insertion of DNA encoding the desired antigenicepitope to produce the fusion protein gene.

The vaccinia promoter and fusion protein gene are then inserted into thevaccinia genome by homologous recombination. This is typically achievedby infecting mammalian cells with vaccinia virus such as the Wyeth (USvaccine) strain and also transfecting the cells with the shuttle vector.The site of insertion is determined by the flanking vaccinia DNAsegments of the shuttle vector. By means of homologous recombination thefunctional TK gene of the wild-type virus is replaced by thenon-functional TK gene sequence included within which is the fusionprotein gene. The resulting recombinant virus is TK⁻ and can thereforebe selected accordingly.

The fusion protein incorporating the antigenic epitope is expressed incells infected with the recombinant viral vector. The fusion protein, itis believed, protrudes from the cell membrane in the manner of normalHA. This enables the antigenic epitope exposed on the surface of the HAto be recognized by the immune system and a suitable immune responsemounted. The viral vectors may therefore by used as vaccines.

The fusion protein itself may alternatively be formulated in a vaccine.The fusion protein is produced by a host and recovered. Cells fromvertebrates or invertebrates, preferably mammalian cells, can be used ashost cell lines. Cell lines such as VERO, HeLa, CHO (Chinese hamsterovary), W138, BHK, COS-7, MDCK and CV-1 may be employed. Expressionvectors for such cells generally contain an origin of replication, apromoter located in front of the gene to be expressed, and any necessaryribosome binding sites, RNA splice sites, polyadenylation site andtranscriptional terminator sequences. Viral promoters preferably areemployed. Viral vectors as above may be used, such as a baculovirusexpression system.

Eucaryotic microbes such as yeast cultures may alternatively by used ashost cells. Saccharomyces cerevisiae strains can therefore be employed.A plasmid vector such as plasmid YRp7 is typically utilised to transformsuch hosts. Any plasmid vector containing a yeast-compatible promoter,origin of replication and termination sequences is suitable.

Vaccines may be administered in any appropriate fashion to a human oranimal. The choice of whether an oral route or a parenteral route suchas sub-cutaneous, intravenous or intramuscular administration is adopteddepends upon the purpose of the vaccination and whether it is a human ormammal being vaccinated. Similar criteria govern the physiologicallyacceptable carrier or diluent employed in the vaccine preparation andthe dose of vaccine.

Both the manner of formulation, the carrier or diluent and the dose forrecombinant viral vaccines may be the conventional ones utilised whenthe unmodified virus is conventionally used as a vaccine. For example,recombinant vaccinia virus may be administered by the dermal route. Aviral vector such as the recombinant vaccinia virus is typicallyadministered, as regards all routes of administration, in an amount of10-1000 ug per dose. More preferably from 10-100 ug of the virus isused. Fusion protein itself may be used as a vaccine in the form of animmunostimulating complex or "iscom" with the matrix of the iscomtypically being the glycoside Quil A (Morein et al, 1984). Fusionprotein may also be administered in an amount of 10-1000 ug per dose,more preferably 10-100 ug per dose, also as regards all routes ofadministration.

The following Example illustrates the invention. In the accompanyingdrawings:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of plasmids pvHAX31 and pvHAX31ΔP;

FIG. 2 shows the plasmid mpsite A, amino acids being shown by theone-letter code;

FIG. 3 shows the replacement of site A of the HA gene by a FMDVΔVP1sequence; and

FIG. 4 shows the FMDVΔVP1 sequences inserted in place of site A in theHA gene, amino acids being shown by the one-letter code.

EXAMPLE

Source of HA

A complete cDNA copy of the HA gene from influenza A/Aichi/2/68 (X31)(Verhoeyen et al, 1980) which had been cloned in M13 mp8 (Messing andVieira, 1982) was obtained from Dr. John Skehel (NIMR, London) (see FIG.1).

Expression of X31 HA using recombinant vaccinia viruses

In order to facilitate transfer of the HA gene to a vaccinia virus (VV)shuttle vector, the above plasmid was cut at the Sma I site within themp8 polylinker, and a Pst I linker was inserted into this site, to formthe clone mpHAX31. This enabled the complete HA gene to be isolated as aPst I fragment.

The VV shuttle vector used was pvp11k (see FIG. 1) which was derivedfrom the vector pH3JΔRIA (Newton et al, 1986) by deletion of extraneousVV sequences. The shuttle vector has a VV promoter (in this case p11k)inserted into the VV thymidine kinase (TK) gene. This vector has aunique Eco RI site immediately following the VV p11k promoter and AUG(Berthelot et al, 1985). To adapt this vector for insertion of the HAgene, the plasmid was cut at the Eco RI site, the sticky-ends filled inusing the Klenow fragment of DNA Polymerase I, and a Pst I linkerinserted to form pvp11k-Pst.

The HA gene was inserted as a Pst I fragment into Pst I-cut,dephosphorylated pvp11k-Pst. Clones with the HA gene in the correctorientation relative to the p11k promoter were identified by restrictionmapping. This clone was designated pvHAX31 (see FIG. 1). In thisconstruction, the natural HA AUG is in the same reading frame as thep11k AUG. Therefore, since there are no in phase stop codons in the HA"untranslated" leader sequence, the expressed HA will be synthesizedwith an additional X amino acids at its N-terminus; these should beremoved during transport and processing of the HA with the HA signalpeptide.

In case the presence of the additional sequences was detrimental tomaturation of the HA, a further construction was prepared. pvHAX31 waspartially digested with Pst I and the sticky ends removed using T4 DNApolymerase (holoenzyme). The linear band was purified on an agarose gel,then re-ligated. Clones were screened for the removal of the correct PstI site by restriction mapping. The correct clone was designatedpvHAX31ΔP. This modification places stop codons in phase with the p11kAUG, and should allow re-initiation at the authentic HA AUG (see FIG.1).

Both these HA constructs were inserted into the genome of the Wyeth (USvaccine) strain of VV, under control of the VV p11k promoter, byhomologous recombination using the flanking TK sequences (Mackett et al,1985 a and b). Individual progeny plaques with a TK⁻ phenotype weretested for expression of HA by reacting methanol--orglutaraldehyde--fixed plaques in situ with rabbit polyclonal anti-HAserum, followed by goat anti-rabbit conjugated with horse radishperoxidase and colour development using 4-chlor-1-naphthol as substrate(Newton et al, 1986) (a "black plaque" assay). Purified plaquesexpressing HA+extra amino acids (vHAX31-J34c) or "natural" HA(vHAX31ΔP-K3) were grown up.

The level of expression of HA was similar in the two constructs (byrough estimation), so re-initiation in vHAX31ΔP was evidently efficient.Also, since the HA with extra N-terminal amino acids was expressed onthe surface of cells infected with vHAX31, transport of this HAevidently unaffected by "junk" sequences at the N-terminus of the signalsequence.

Replacement of HA antigenic site A with FMDV sequences

In all experiments replacement of site A has been done using the pvHAX31construct (i.e. with N-terminal "junk" amino acids). This plasmid waspartially digested with HindIII and the sticky ends filled in as above.Linear plasmid was isolated and religated. Clones were screened for theremoval of the HindIII site located at the 5' end of the VV TK gene; thecorrect clone was designated pvHAΔH. This clone now had a unique HindIIIsite located within the HAv gene 5' of site A.

To facilitate manipulation of site A, pvHAΔH was cut off the uniqueHindIII and HincII sites, and the 180b fragment containing site A wassubcloned into M13 mp11. The large fragment was also isolated for lateruse. Correct clones were identified by sequencing picked plaques; thisclone was designated mpsite A (see FIG. 2). RF DNA from mpsiteA was cutwith HindIII and AccI (the M13 polylinker AccI site was removed in theabove construction, leaving only the HA AccI site at the 3' side of siteA) and the 165 bp fragment purified on an agarose gel. The largefragment was also isolated for later use. This fragment was then cutwith AvaII and the 145 bp HindIII-AvaII fragment purified on anacrylamide gel. This removes the HA site A sequences (FIG. 3).

Complementary synthetic oligonucleotides coding for FMDV sequences wereprepared--these were constructed so that when annealed an AvaII overhangwas left at the 5' end and an AccI overhang at the 3' end. Theseannealed oligonucleotides, the HA Hind III-AvaII fragment and the largeHindIII-I fragment from mpsiteA were joined in a three-way ligation.Picked M13 plaques were sequenced to identify clones carrying FMDVsequences and to check the fidelity of the ologonucleotide synthesis andcloning, so as to ensure that the FMDV sequences were in the correctreading frame with the HA at both ends. RF DNA's from the variousmodified mpHA clones were cut with HindIII and HincII and the modified180-200 bp fragments purified. These fragments were then re-cloned intothe HindIII-HincII cut preparation of pvHAΔH from above. The portions ofthe FMDV immunogenic site which have been inserted into HA site A areshown in FIG. 4.

The modified HAΔ (pvHA-80/81, pvHA-82/83, pvHA-122/124) were introducedinto the VV genome by homologous recombination using flanking TKsequences, and plaque-picked TK⁻ virus analysed. Viruses were initiallyscreened for their ability to react with polyclonal anti-HA serum inblack plaque assays of methanol--and glutataldehyde--fixed plaques. Allthree modified HAs were able to react with the polyclonal antiserum, andwere expressed on the surface of infected cells. The recombinant VVswere then tested for their ability to react with anti-VP1₁₄₁₋₁₆₀antiserum using glutataldehyde-fixed plaques. Again, all three modifiedHAs were positive with the anti-peptide serum, while the unmodifiedrecombinant (vHAX31-J34c) was not.

The protein species synthesized in CV-1 cells infected withvHAX31(J34c), vHA80/81(Q3/1), vHA82/83(S6/1) and vHA122/124(T3/1) wereexamined. Infected cells were labelled with ³⁵ S methionine andcytoplasmic extracts prepared. The extracts were immune-precipitatedusing polyclonal anti-HA serum and protein A, and the precipitatesexamined by SDS-PAGE (Laemmli, 1970). In all cases, a protein speciescorresponding to HA₀ (uncleaved HA) was observed. The species was thesame size in all cases, but occasionally a slightly higher mol. wt. bandwas observed; this may be due to inefficient removal of the signalpeptide caused by the extra "junk" amino acids. In some experiments,where a long labelling period was used, there was some processing of HA₀into HA1 and HA2, which co-migrated with those species extracted frompurified X31 virus particles. vHA122/124(T3/1) was deposited at theEuropean Collection of Animal Cell Cultures, Porton Down, GB on Aug. 25,1989 under Accession No. V89082504.

REFERENCES

Bertholet et al (1985) PNAS 82 2096

Gething and Sambrook (1982) Nature 300 598-625

Laemmli (1970) Nature 227 680-685

Mackett et al (1985a) DNA Cloning, A Practical Approach (ed. D. M.Glover) 2 191 IRL Press Oxford

Mackett et al (1985b) Techniques--in Gene Cloning Vol. 2

Messing and Vieira (1982) Gene 19 269-276

Morein et al (1984) Nature 308 457-460

Newton et al (1986) Vaccines 86: New Approaches to Immunization, ColdSpring Harbor Laboratory, 303-309

Smith et al (1983) PNAS 80 7155-59

Verhoeyen et al (1980) Nature 286 771-776

Wiley et al (1981) Nature 289 373-378

Wilson et al (1981) Nature 289 366-373

We claim:
 1. A DNA sequence encoding a fusion protein which comprisesinfluenza virus haemagglutinin (HA) and, at antigenic site A of HA, aheterologous epitope, wherein all or part of said antigenic site isreplaced by said epitope and said protein presents said epitope in amanner recognizable by an immune system.
 2. The DNA sequence accordingto claim 1, wherein said epitope is selected from the group consistingof an epitope of a virus, an epitope of a bacterium and an epitope of aprotozoan.
 3. The DNA sequence according to claim 2, wherein saidepitope is selected from the group consisting of an epitope offoot-and-mouth disease virus, an epitope of poliovirus, an epitope ofhuman rhinovirus and an epitope of hepatitis B virus.
 4. The DNAsequence according to claim 2, wherein said epitope is an epitope ofPlasmodium falciparum.
 5. A eucaryotic expression vector comprising aDNA sequence encoding a fusion protein, which protein comprisesinfluenza virus haemagglutinin (HA) and, at antigenic site A of HA, aheterologous epitope, wherein all or part of said antigenic site isreplaced by said epitope and said protein presents said epitope in amanner recognizable by an immune system.
 6. The vector according toclaim 5, wherein said epitope is selected from the group consisting ofan epitope of a virus, an epitope of a bacterium and an epitope of aprotozoan.
 7. The vector according to claim 5, wherein said vector is aviral vector.
 8. The vector according to claim 6, wherein said epitopeis selected from the group consisting of an epitope of foot-and-mouthdisease virus, an epitope of poliovirus, an epitope of human rhinovirusand an epitope of hepatitis B virus.
 9. The vector according to claim 6,wherein said epitope is an epitope of Plasmodium falciparum.
 10. Thevector according to claim 7, wherein said vector is a recombinantvaccinia virus that comprises said DNA sequence.
 11. A eucaryotic hostcell comprising a vector comprising a DNA sequence encoding a fusionprotein, which protein comprises influenza virus haemagglutinin (HA)and, at antigenic site A of HA, a heterologous epitope, wherein all orpart of said antigenic site is replaced by said epitope and said hostcell expresses said fusion protein on the surface thereof and presentssaid epitope in a manner recognizable by an immune system.
 12. The hostaccording to claim 11, wherein said epitope is selected from the groupconsisting of an epitope of a virus, an epitope of a bacterium and anepitope of a protozoan.
 13. The host according to claim 11, wherein saidepitope is an epitope of Plasmodium falciparum.
 14. The host accordingto claim 11 wherein said host is a mammalian cell.
 15. A host accordingto claim 12, wherein said epitope is selected from the group consistingof an epitope of foot-and-mouth disease virus, an epitope of poliovirus,an epitope of human rhinovirus and an epitope of hepatitis B virus. 16.A eucaryotic plasmid expression vector compatible with a yeast hostcomprising a DNA sequence encoding a fusion protein, which proteincomprises influenza virus haemagglutinin (HA) and, at antigenic site Aof HA, a heterologous epitope, wherein all or part of said antigenicsite is replaced by said epitope and said protein presents said epitopein a manner recognizable by an immune system.
 17. A yeast hostcomprising a vector compatible with said host, comprising a DNA sequenceencoding a fusion protein, which protein comprises influenza virushaemagglutinin (HA) and, at antigenic site A of HA, a heterologousepitope, wherein all or part of said antigenic site is replaced by saidepitope and said protein presents said epitope in a manner recognizableby an immune system, and wherein said host expresses said fusionprotein.